The hypothesis to be tested is that sustained production of free radicals by mitochondria leads to critical mitochondrial DNA damage and dysfunction that are amplified to lethal cellular events by initiating apoptotic pathways through cyctochrome c release and c-jun N-terminal kinase activation. The four specific aims are: (1) To determine the effects of age on the regulation of mitochondrial functions by metabolic state, oxygen, and nitric oxide. (2) To identify the mechanisms and consequences of mitochondrial DNA (mtDNA) oxidative damage leading to specific mtDNA mutations. (3) To elucidate the consequences of mtDNA mutations at the cellular level within the framework of mitochondrial changes signaling for apoptosis. (4) To examine the role of the thiol/disulfide status in mtDNA damage and further consequences at mitochondrial and cellular levels. The focus of this research proposal is to identify the critical molecular events that are involved in the sequence from mitochondrial dysfunction to apoptosis by assessing various stimulatory and protective pathways that can initiate or delay the onset of cell death.